The invention relates to assays for nucleoside diphosphates, particularly ADP and GDP, and assays for nucleoside triphosphates, particularly ATP and GTP.
Nucleoside diphosphates and triphosphates play important roles in biology. ADP is the immediate precursor for the formation of ATP, the universal currency of cellular energy. GDP is a substrate for succinyl CoA synthetase, a key enzyme of the Krebs cycle, and is formed during gluconeogenesis by phosphoenolpyruvate carboxykinase. It is also essential in G-protein signalling, microtubule growth, and visual excitation. UDP is involved in the epimerisation of galactose to glucose, the formation of sucrose, and in the growth of glycogen. CDP is an important group in the synthesis of phosphoglycerides. Nucleoside diphosphates are also the products of reactions catalysed by several major classes of enzymes, such as triphosphatases and kinases, and are therefore produced by many cellular processes, including motility, muscle contraction, DNA synthesis, transcription, translation and nitrogen fixation.
The detection and measurement of nucleoside diphosphates and triphosphates is thus important in the study of biology and metabolism, particularly in bioenergetics.
Assays for ADP and ATP in biological samples based on luciferase have been known for over 20 years [e.g. refs 1, 2, 3, 4]. Bioluminescent assays for ADP and ATP have been described for use in muscle and adipose tissue biopsies [5] and a three-enzyme bioluminescent system utilising luciferase has been reported for use in bacterial cell extracts [6]. A bioluminescent ADP assay optimised for use at high ATP:ADP ratios has been reported [7], but this requires the enzymatic removal of ATP. In general, it is easier to measure ATP in the presence of ADP than to measure ADP in the presence of ATP.
Enzymatic spectrophotometric assays have also been described [e.g. 8].
Assays for GDP and GTP in biological samples are also well known [e.g., refs 9 and 10].
Reference 11 discloses column-based chromatographic assays for GDP, CDP and UDP. Radioactive assays for GDP and GTP have also been described [12, 13]. NMR-based assays for measuring in vivo ADP levels are known for yeast [14], and NMR has also been used to measure ADP and ATP and erythrocytes [15].
According to the present invention, nucleoside diphosphates are detected or measured by following the dephosphorylation of the phosphoenzyme form of nucleoside diphosphate kinase (NDPK), and nucleoside triphosphates are detected or measured by following the phosphorylation of NDPK to its phosphoenzyme form.
The invention thus provides (a) a process for detecting the presence of a nucleoside diphosphate in a sample, comprising the step of detecting the dephosphorylation of the phosphoenzyme form of a nucleoside diphosphate kinase, and (b) a process for detecting the presence of a nucleoside triphosphate in a sample, comprising the step of detecting the phosphorylation a nucleoside diphosphate kinase to the phosphoenzyme form.
The process will typically comprise the steps of:
causing nucleoside diphosphate in sample to bind to NDPK phosphoenzyme, or causing nucleoside triphosphate in sample to phosphorylate NDPK; and
detecting a change in a characteristic of the enzyme which differs between its phosphorylated and unphosphorylated forms.
The term xe2x80x9cNDPKxe2x80x9d means an enzyme having the activity of the enzyme as EC 2.7.4.6, namely the transfer of the xcex3-phosphate group of a nucleoside triphosphate (N1TP) to a nucleoside diphosphate (N2DP) via a pin-pong mechanism:
N1TP+N2DPxe2x86x92N1DP+N2TP
Based on this reaction scheme, the systematic name of NDPK is xe2x80x9cATP:nucleoside-diphosphate phosphotransferasexe2x80x9d, but the common name is xe2x80x9cnucleoside diphosphate kinasexe2x80x9d. The enzyme has also been variously described as kinase (phosphorylating), nucleoside diphosphate; nucleoside 5xe2x80x2-diphosphate kinase: nucleoside diphosphate (UDP) kinase: nucleoside diphosphokinase: nucleotide phosphate kinase: NM23.
NDPKs have been described for a number of organisms, both prokaryotic and eukaryotic e.g. human, cows, monkeys, mice. Xenopus, oats, peas, potatoes, yeast, Bacillus subtilis, E. coli, Myxococcus xanthus, avian myeloblastosis virus etc. These differ by cellular location, molecular weight, oligomeric structure, isoelectric point, reaction kinetics, substrate preference, pH optimum, pH range, temperature optimum, cation requirements (Mn2+, Mg2xe2x88x92, Co2+, Ca2 etc.), and various isoforms have been described. Given the variety of suitable enzymes available, the skilled person can easily select and purify a NDPK to suit any particular situation.
The NDPK enzyme uses a ping-pong mechanism, transferring the xcex3-phosphate from a nucleoside triphosphate (N1TP) to an active site histidine to form a phosphoenzyme intermediate, and then to a nucleoside diphosphate (N2DP). The invention is based on the finding that the phosphoenzyme intermediate is stable over a time-scale that allows its detection and measurement. Other enzymes that phosphorylate nucleoside diphosphates via a phosphoenzyme intermediate, preferably with a single binding site for nucleotide, may also be used in the invention.
The phosphoenzyme is able to transfer its phosphate group to N2DP in a sample to form the corresponding N2TP. Detection of this transfer can therefore be used for the detection of nucleoside diphosphate. To detect nucleoside diphosphate according to the invention, therefore, phosphoenzyme is required as a reagent. This can be readily formed by, for example, incubating NDPK with excess NTP, typically ATP. Formation of phosphoenzyme in this way is facilitated by removing Mg2+ [16], for instance by using EDTA. Chemical phosphorylation of histidine using phosphormamide as a phosphorylating agent may also be used [17].
The phosphoenzyme can be isolated for use as a reagent. It has been found that the phosphoenzyme can be stored on ice for over 48 hours without dephosphorylation, and can be stored for longer periods (at least 5 months) at xe2x88x9280xc2x0 C. (although repeated freeze-thawing results in some dephosphorylation). The stability of the phosphoenzyme over the time range needed for its preparation, and subsequently for monitoring kinetic events such as the release of ADP from an ATPase, is particularly advantageous.
When added to a sample containing NDP, the phosphoenzyme is dephosphorylated by the transfer of its phosphate group to the NDP. When added to a sample containing NTP, the phosphoenzyme is formed by the transfer of the NTP xcex3-phosphate group to the enzyme. The invention relies on the ability to distinguish between the phosphorylated and dephosphorylated forms of NDPK.
In order to distinguish the phosphorylated and unphosphorylated forms of NDPK, any suitable measurable change can be used.
For instance, intrinsic properties of the enzyme can be used. Depending on the particular NDPK chosen, the following methods are examples of how dephosphorylation/phosphorylation may be detected, with varying levels of sensitivity:
The location of a phosphate (i.e. either bound to NDPK, or as the xcex3-phosphate of a NTP) can be ascertained by following the 31P NMR spectrum.
Protons whose environment changes upon dephosphorylation can be detected by, for instance NMR.
Dephosphorylation may cause a change in the fluorescence of a tryptophan residue in the protein [e.g., ref 18].
Dephosphorylation can be detected by following the loss of 32P from radio-labelled phosphoenzyme. The radio-isotope can be conveniently incorporated into NDPK by using [xcex3-32P]ATP.
Circular dichroism, or any other suitable spectrometric technique, can detect conformational changes which occur on dephosphorylation.
Dephosphorylation may result in a change in surface plasmon resonance properties.
Rather than using properties inherent in the wild-type enzyme, it may be desired to modify the enzyme in some way. This may also be important where dephosphorylation of the NDPK of choice does not exhibit an intrinsic measurable change which can be readily followed.
One particularly preferred modification is the addition of a fluorescent label to the enzyme, typically via a cysteine residue. If the wild-type protein lacks a suitable cysteine residue e.g. the NDPK of Myxococcus xanthus (SEQ ID NO: 1), this can easily be introduced by mutagenesis [e.g. 19]. A suitable position for mutation can easily be determined by the skilled person, whilst ensuring that the mutation does not disrupt the enzymatic activity [e.g. 20]. At any given amino acid residue, particular labels may give better results than others. Suitable combinations of label and residue can be determined by routine experimentation.
Preferred fluorescent labels are based around coumarin. Particularly preferred is N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide [21: FIG. 1], referred to simply as xe2x80x98IDCCxe2x80x99 hereafter. This is preferably attached to a cysteine residue, and preferably exhibits a high fluorescence when NDPK is phosphorylated, and a low fluorescence when NDPK is dephosphorylated. When suitably attached to NDPK, this label offers the advantage that the phosphoenzyme can detect small quantities of ADP in the presence of much higher concentrations of ATP. This is extremely important for experiments in situations where ATP levels are high e.g. in single muscle fibres. It is also able to respond very quickly to changes in ADP levels, and gives a large signal change over a range of several hundred micromolar.
Other labels which can be introduced in similar ways include ESR labels, luminescent labels, phosphorescent labels, and other suitable chromophores.
It will be appreciated that some of the various options available to the skilled person are more suitable than others for detecting the phosphorylation/dephosphorylation of the enzyme in real-time. Fluorescence is highly suitable for real-time detection, whereas methods such as those using radio-labels are more suitable for measuring end-points.
The nucleoside diphosphate/triphosphate which is assayed must be a substrate of the NDPK being utilized. Various NDP substrates have been described [e.g. 22, 23, 24] including ADP, CDP, GDP, UDP, IDP, XDP, their deoxy-derivatives (e.g. dADP, dCDP, dGDP, dTDP, dUDP). 6-aza-UDP, 8-bromo-IDP, 8-aza-GDP, and 8-aza-UDP, and adenosine 5xe2x80x2-methylene diphosphate. Each of these compounds is phosphorylated by the phosphoenzyme (with varying reaction affinities and kinetics, depending on both the NDPK and the substrate being utilised), and can thus be assayed according to the invention.
The invention is preferably used to detect and measure ADP or GDP. Accordingly, a NDPK may be chosen which shows a preference towards one of these substrates.
In preferred embodiments of the invention, the detection process gives quantitative data, that is to say the invention provides a process for quantifying nucleoside diphosphate or triphosphate in a sample. This will typically involve the step of relating a change in the detectable characteristics of a NDPK to a concentration of NDP or NTP. It will be appreciated that this may require a calibration to be performed (e.g. for measuring dephosphorylation via a fluorescent label such as IDDC) or comparison with a standard. Calibration will typically be performed for the desired range of concentrations to be measured.
In a first quantitative aspect, the amount of nucleoside diphosphate or nucleoside triphosphate is determined by measuring the decrease (NDP) or increase (NTP) in the level of phosphoenzyme after the addition of phosphoenzyme (NDP) or unphosphorylated enzyme (NTP) to a sample.
In a second quantitative aspect, the rate of production of nucleoside diphosphate or triphosphate can be determined by following the decrease (NDP) or increase (NTP) in the level of phosphoenzyme over time. By fitting the measured values to a suitable mathematical model (e.g. a simple model based on first-order exponential decrease), the rate of nucleoside diphosphate or triphosphate production can be determined. In this aspect, dephosphorylation of the phosphoenzyme (NDP) or phosphorylation of the enzyme (NTP) is preferably measured using a real-time detection method.
The process of the invention is preferably suitable for use either in vivo or vitro. The method is preferably suitable for in situ use in a muscle fibre, and is preferably gives data suitable for calculating the rate of ADP release from actomyosin.
As well as the step of detecting the dephosphorylation of the phosphoenzyme (NDP-related aspects), the process will usually comprise the initial step of adding NDPK phosphoenzyme to a sample of interest. This may be preceded by the preparation of phosphoenzyme from unphosphorylated NDPK.
The process may also include a step of analysing any data obtained during the process, such as fitting the data to an equation in order to derive quantitative values.
The process preferably avoids the use of reagents such as theophylline, desdanine and Ag+, which may inhibit the NDPK activity.
As well as the above processes, the invention provides reagents for use in the processes.
The invention provides NDPK which is modified to carry a label which gives a different detectable signal when the enzyme is phosphorylated from when it is unphosphorylated.
The label on the modified NDPK may be a fluorescent group, preferably IDCC.
The label will typically be attached to an amino acid residue in the enzyme. It is preferred to attach the label to a cysteine residue.
A particularly preferred reagent is the NDPK of M. xanthus carrying a Asp112xe2x86x92Cys mutation, and carrying an IDCC label at this mutated residue. This reagent as a phosphoenzyme is about three orders of magnitude more sensitive to ADP than to ATP.
The invention also provides a NDPK modified by the attachment of at least one detectable label that is sensitive to the binding of a nucleoside diphosphate.
The invention also provides substrates having these NDPK reagents immobilised thereto. These include columns or beads. This may be used in combination with 32P-phosphoenzyme, such that ADP in a sample incubated with immobilised NDPK will become radio-labelled in its conversion to ATP. Radioactivity in free solution will therefore indicate the amount of ADP in the original sample.
The invention also provides processes for the production of these NDPK reagents.
Furthermore, the invention provides these NDPK reagents for use as in vivo or in vitro diagnostic reagent.